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Vansteelandt M - Kerzaon I - Blanchet E - Robiou Du Pont T - Joubert Y - Monteau F - Le Bizec B - Frisvad JC - Pouchus YF - Grovel O
Patulin and secondary metabolite production by marine-derived Penicillium strains
Fungal Biology
Genus Penicillium represents an important fungal group regarding to its mycotoxin production. Secondary metabolomes of eight marine-derived strains belonging to subgenera Furcatum and Penicillium were investigated using dereplication by liquid chromatography (LC)eDiode Array Detector (DAD)emass spectrometry (MS)/MS. Each strain was grown on six different culture media to enhance the number of observable metabolites. Thirty-two secondary metabolites were detected in crude extracts with twenty first observations for studied species. Patulin, a major mycotoxin, was classically detected in extracts of Penicillium expansum, and was also isolated from Penicillium antarcticum cultures, whose secondary metabolome is still to be done. These detections constituted the first descriptions of patulin in marine strains of Penicillium, highlighting the risk for shellfish and their consumers due to the presence of these fungi in shellfish farming areas. Patulin induced acute neurotoxicity on Diptera larvae, indicating the interest of this bioassay as an additional tool for detection of this major mycotoxin in crude extracts. © 2012 The British Mycological Society. Published by Elsevier Ltd.

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Le Bizec B
Editorial - Brazilian Ministry of Agriculture, Livestock and Food Supply (MAPA): strategies to tackle chemical food safety issues
Food Additive and Contaminants

Sous presse, 2012
DOI 10.1007/s00216-012-5739-7
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Vanden Bussche J - Sterk S - De Brabander H - Blokland MH - Deceuninck Y - Le Bizec B - Vanhaecke L
Thyreostatic drugs, stability in bovine and porcine urine
Analytical and Bioanalytical Chemistry
Thyreostatic drugs, illegally administrated to livestock for fattening purposes, are banned in the European Union since 1981. For monitoring their illegal use, sensitive and specific analytical methods are required. In this context, the knowledge of the stability in a matrix is of primary importance. This study aimed at evaluating the effects of preservation, number of freeze–thaw cycles, and matrixrelated variables on the stability of thyreostatic drugs in the urine of livestock. Finally, the developed conservation approach was applied on incurred urine samples, which displayed traces of the thyreostat thiouracil below the recommended concentration of 10 μg L-1. The stability study confirmed the negative influence of preservation (8 h) at room temperature and at -70 °C, decreases in concentration of more than 78.0% were observed for all thyreostats, except for 1-methyl-2-mercaptoimidazole and 2-mercaptobenzimidazole. Additionally, investigation of matrix-related variables indicated significant impacts of the presence of copper (p=0.001) and the pH (p=0.002). Next, an optimised pretreatment (pH 1 and 0.1 M ethylenediaminetetraacetic acid disodium salt dehydrate) significantly differing from the original conservation approach (p<0.05) was developed, which proved capable of delaying the decrease in concentration and improved the detection in time for both spiked as well as incurred urine samples. In the future, it seems highly advisable to apply the developed pre-treatment on incurred urines upon sampling, before thyreostat analysis. Additionally, it is recommendable to limit preservation of urine samples at room temperature, but also in the freezer prior to thyreostat analysis. © Springer-Verlag 2012

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Marteau C - Chevolleau S - Jouanin I - Perdu E - De Sousa G - Rahmani R - Antignac JP - Le Bizec B - Zalko D - Debrauwer L
Development of a LC-APPI-HRMS analytical strategy for the simultaneous analysis of PBDEs, OH-PBDEs and other PBDE metabolites. Application to BDE-47 metabolism in human hepatocytes
Rapid Communication in Mass Spectrometry
Polybrominated diphenyl ethers (PBDEs) are flame retardants widely used in electronic and domestic goods. These persistent pollutants are present in the environment and in humans, and their toxicological properties are of growing concern. PBDEs can be metabolised into compounds suspected to be responsible for their toxicity. These metabolites have been characterised quite well in rodents and fish, but available information in humans remains scarce. For their identification, an efficient method for the simultaneous analysis of PBDEs, hydroxylated PBDEs (OH-PBDEs), and other PBDE metabolites in a single run was needed and has been developed in this work. Atmospheric pressure ionisation modes were compared, and Atmospheric Pressure Photo-Ionization (APPI) was selected. After careful setting of APPI parameters such as dopant and operating temperature, the optimised method was based on APPI ionization coupled to High-Resolution Mass Spectrometry operating in the full scan mode at a resolution of 60 000. This provided excellent sensitivity and specificity, allowing the discrimination of signals which could not be resolved on a triple quadrupole used as a reference. The full-scan high-resolution acquisition mode allowed monitoring of both parent PBDEs and their metabolites, including hydroxylated PBDEs, with detection limits ranging from 0.1 pg to 4.5 pg injected on-column based on the investigated standard compounds. The method was applied to the study of BDE-47 metabolism after incubation with human primary cultures of hepatocytes, and proved to be efficient not only for monitoring the parent compound and expected hydroxylated metabolites, but also for the identification of other non-targeted metabolites. In addition to hydroxy-BDE-47, several conjugated metabolites could be located, and the formation of a dihydrodiol derivative was evidenced for the first time in the case of PBDEs in this work. Copyright © 2012 John Wiley & Sons, Ltd.

2012, 26, 819–827
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Bichon E - Béasse A - Prevost S - Christien S - Courant F - Monteau F - Le Bizec B
Improvement of estradiol esters monitoring in bovine hair by dansylation and LC-MS/MS analysis in MRM and precursor ion scan modes
Rapid Communications in Mass Spectrometry
RATIONALE: The control of forbidden anabolic practices in cattle in the European Union has become challenging since endogenous compounds such as estradiol derivatives can potentially be used as growth promoters. Due to the great difficulty in establishing a reference threshold value for endogenous steroids, the direct detection of steroid esters in hair is an efficient strategy for the detection of ’natural’ steroid abuse in cattle. METHODS: The present study aimed to develop and validate according to the current European standards a specific liquid chromatography/tandem mass spectrometry (LC/MS/MS) analytical strategy to monitor estrogen esters in bovine hair. The analysis was performed by positive ion electrospray ionisation (ESI+) after dansylation. Two acquisition modes were then assessed: single reaction monitoring and precursor ion scanning. RESULTS: The results showed that the introduction of a dansylation step strongly improves the sensitivity of the detection of estradiol-17-esters by LC/(ESI+)-MS/MS. The CCa values are in the range 1–10 ng g-1 after optimisation, except for estradiol decanoate for which the derivatisation is not efficient. In addition, this LC/MS/MS approach makes it possible to carry out a precursor ion scan to screen for the presence of these estradiol 17-esters in hair samples. CONCLUSIONS: Based on the specific product ions, i.e. m/z 255 in native conditions or m/z 171 after dansylation, this strategy has the advantage of detecting any (un)known estradiol ester and of giving access to the [M +H]+ ion of the suspected ester through only a single analysis. Copyright © 2012 John Wiley & Sons, Ltd.

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Anizan S - Bichon E - Monteau F - Cesbron N - Antignac JP - Le Bizec B
Gas chromatography coupled to mass spectrometry based metabolomic to screen anabolic practice in cattle
Journal of Mass Spectrometry
The use of anabolic steroids as growth promoters for meat-producing animals is banned within the European Union. However, screening for the illegal use of natural steroid hormones still represents a difficult challenge because of the high interindividual and physiological variability of the endogenous concentration levels in animals. In this context, the development of untargeted profiling approaches for identifying new relevant biomarkers of exposure and/or effect has been emerging for a couple of years. The present study deals with an untargeted metabolomics approach on the basis of GC-MS aiming to reveal potential biomarkers signing a fraudulent administration of 4-androstenedione (AED), an anabolic androgenic steroid chosen as template. After a sample preparation based on microextraction by packed sorbent, urinary profiles of the free and deglucurono-conjugates urinary metabolites were acquired by GC-MS in the full-scan acquisitionmode. Data processing and chemometric procedures highlighted 125 ions, allowing discrimination between samples collected before and after an administration of 4-AED. After a first evaluation of the signal robustness using additional and independent non-compliant samples, 17 steroid-like metabolites were pointed out as relevant candidate biomarkers. All these metabolites were then monitored using a targeted GC-MS/MS method for an additional assessment of their capacity to be used as biomarkers. Finally, two steroids, namely 5α-androstane-3β,17α-diol and 5α-androst-2-en-17-one, were concluded to be compatible with such a definition and which could be finally usable for screening purpose of AED abuse in cattle. Copyright © 2012 John Wiley & Sons, Ltd.

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Desdoits C - Albert O - Le Bizec B - Lesné L - Cravédi JP - Zalko D - Courant F - Guillé F - Dejucq-Rainsford N - Jégou B
Di (2-ethylhexyl) phthalate and mono-(2-ethylhexyl) phthalate are anti-androgenic in the adult human testis
Human reproduction
background: Phthalic acid esters are widely used in the manufacture of plastics. Numerous studies have shown that these phthalates impair testicular testosterone production in the rat. However, the scarce and contradictory data concerning humans have cast doubt over whether these compounds are also anti-androgenic in man. We therefore investigated the direct effects of di-(2-ethylhexyl) phthalate (DEHP) and mono-(2-ethylhexyl) phthalate (MEHP) on organo-cultured adult human testis and a human cell line. methods: Adult human testis explants or NCI-H295R adrenocortical human cells were cultured with DEHP or MEHP. The effects of ketoconazole, used as a reference molecule, were also assessed. results: In both models, DEHP and MEHP significantly inhibited testosterone production. The effects of both phthalates appeared to be specific for steroidogenesis, as INSL3 production by Leydig cells was not altered. Furthermore, the phthalates of interest had no effect on inhibin B production by Sertoli cells or on germ cell apoptosis. As only a small fraction of the phthalates added was found in the testis explants, and as these compounds were found to be metabolized, we estimate that the anti-androgenic effects observed occurred at concentrations of phthalates that are of the same order of magnitude as exposures reported in the literature for men. conclusions: We provide the first evidence that DEHP and MEHP can inhibit testosterone production in the adult human testis. This is consistent with recent epidemiological findings of an inverse correlation between exposure to MEHP and testosterone concentrations. © The Author 2012. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.

Sous presse, 2013
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Rochereau-Roulet S - Gicquiau A - Morvan ML - Blanc G - Dervilly-Pinel G - Le Bizec B
Recombinant bovine growth hormone identification and kinetic of elimination in treated rainbow trouts
Food Additives and Contaminants

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Miege C - Roy A - Labadie P - Budzinski H - Le Bizec B - Veyrand B - Vorkamp K - Persat H - Coquery M - Babut M
Occurrence of priority and emerging organic compounds in fishes from the Rhone River (France)
Analytical and Bioanalytical Chemistry
The main objective of this study was to collect new data on the occurrence, levels of priority and emerging organic compounds in freshwater fish sampled in the Rhone River. The 34 studied contaminants included alkylphenols, bisphenol A, polybromodiphenylethers (PBDE), perfluorinated compounds, hexabromocyclododecanes (HBCD), hexachlorobenzene and hexachlorobutadiene (HCBD). About 50 fish samples (individual specimens or pooled fish) were collected from three sites located upstream and downstream of the Lyon metropolitan area in the Rhone River (France). Four species were caught at each site, namely: the barbel (Barbus barbus), the common bream (Abramis brama), the white bream (Blicca bjoerkna) and the chub (Squalius cephalus). Some contaminants were quantified in all the 32 fish samples analysed: 4-nonylphenol, α-HBCD, the six PBDE congeners (28, 47, 99, 100, 153, 154), perfluorooctanesulfonate (PFOS) and perfluorodecanoic acid. Twenty three of the 32 samples had a concentration of PFOS above the Environmental Quality Standards (EQS) (up to six times higher than the EQS), and all the 32 samples had concentrations of PBDE above the EQS (up to 4,000 times higher, with the sum of six PBDE varying from 4.5 to 182 ng/g dry weight). Clearly, the interest to consider PFOS and HBCD as new priority substances is confirmed. In contrast, the pertinence of a priority status for HCBD, which was never quantified in our study, might have to be reconsidered in the future. © Springer-Verlag 2012

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Lambert O - Veyrand B - Le Bizec B - Antignac JP - Marchand P - Piroux M - Puyo S - Thorin C - Delbac F - Pouliquen H
Polycyclic aromatic hydrocarbons: bees, honey and pollen as sentinels for environmental chemical contaminants
Three beehive matrices, sampled in six different apiaries from West France, were analyzed for the presence of four polycyclic aromatic hydrocarbons (PAH4: benzo[a]pyrene, benzo[a]anthracene, benzo[b]fluoranthene and chrysene). Samples were collected during four different periods in both 2008 and 2009. Honey samples showed the lowest levels of PAH4 contamination (min = 0.03 µg kg-1;max = 5.80 µg kg-1; mean = 0.82 µg kg-1; Sd = 1.17). Bee samples exhibited higher levels of PAH4 contamination (min = 0.32 µg kg-1; max = 73.83 µg kg-1; mean = 7.03 µg kg-1; Sd = 17.83) with a great dispersion of the concentrations due to four main events of high concentrations. Pollen samples showed only one major episode with the highest PAH4 concentration found (min = 0.33 µg kg-1; max = 129.41 µg kg-1; mean = 7.10 µg kg-1; Sd = 22.28). The PAH4 concentrations found were significantly influenced by the landscape context for all beehive samples. Crown Copyright © 2011 Published by Elsevier Ltd. All rights reserved.

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